Synthesis and Evaluation of Near-Infrared (NIR) Dye−Herceptin Conjugates as Photoacoustic Computed Tomography (PCT) Probes for HER2 Expression in Breast Cancer

Abstract

We are evaluating PCT imaging in conjunction with NIR-dye labeled Herceptin antibody for noninvasive assessment of HER2 expression in tumors. Herceptin was labeled with Alexa Fluor-750 amine reactive dye for characterization of photoacoustic and fluorescence signals. Measurements were performed in solution and after incubation in cultured cell lines that were positive or negative in expression of HER2. The dye to antibody ratio was controlled to achieve a broad range of degree of labeling (DOL = 2 to 15). Photoacoustic signal intensity of Herceptin-dye conjugates in solution increased with increasing over the entire DOL range studied. In contrast, fluorescence exhibited significant quenching for higher DOL. In vitro PCT imaging of the labeled HER2 (+) and HER2 (−) cells revealed the targeting specificity of the NIR-dye labeled Herceptin. In HER2 (+) cells lines, photoacoustic signal intensity gradually increased with increasing DOL and with increasing number of cells. These results demonstrate that PCT-based measurement of HER2 receptor binding using NIR-dye labeled Herceptin is feasible. The absence of a quenching effect with increased DOL advantages this method over traditional methods based on fluorescence measurement.

Synthesis and biological evaluation of panitumumab–IRDye800 conjugate as a fluorescence imaging probe for EGFR-expressing cancers

“…In the bioconjugation reaction the succinimidyl ester group of NIR-dye reacts with a lysine residue of the mAb to form a stable peptide linkage. 15 After 2 h of reaction, the conjugate was purified on a G25 size exclusion column and the product was collected in fractions (Figure 1). Early-front and late-tail fractions from the column (Figure 1) were discarded due to low protein concentration and possible mixing of free dye into the conjugate.…”

Micro-CT enables microlocalisation and quantification of Her2-targeted gold nanoparticles within tumour regions

“…In other non-CT work, Herceptin has been used for targeted imaging using radioactivity [22], radioactive liposomes [18], magnetic nanoparticles [24], gadolinium [24][25][26] 18 F-PET [27], fluorophores [28][29][30] and near-infrared probes [28,31,32]. Consistent with these studies, we found a tumour uptake value (8.3% id g -1 ) similar to these other Herceptin adducts, indicating that the 15 nm AuNP-antibody conjugate delivery does not substantially alter the tumour uptake compared with, for example, a radiolabelled Herceptin antibody targeted to BT474 tumours, which showed 7.8% id g -1 localisation [22].…”

Biomedical Applications of Photoacoustic Imaging with Exogenous Contrast Agents

“…There are many bio-compatible dyes that absorb in the optical window, including Indocyanine Green (ICG), 44 IRDye800CW, 101 AlexaFluor 750, 5 and methylene blue, 99 which all have been used in PA imaging. These dyes are typically small molecules-on the order of 1 nm-and are able to quickly clear the body through the renal system.…”

“…In addition to the ICG PEBBLEs, dyes can be directly conjugated to molecular targeting agents. 5,101 Furthermore, a fluorescent activatable molecular probe named MMPSense 680 can also be used for molecular imaging. While in its inactivated state, it does not fluoresce, but, when activated by a matrix metalloproteinase (MMP), the agent fluoresces and its absorption spectrum changes, producing a unique PA signature.…”